The nervous system, especially the brain, is one of the most complex tissues composed of numerous cell types and subtypes organized with delicate topological characteristics, presenting unique challenges to traditional gene expression analysis techniques. The RNAscope®in situ hybridization assay uniquely addresses these challenges by enabling highly sensitive and specific gene expression analysis at the single-transcript detection level and single-cell resolution, while preserving spatial and morphological context. In particular, the multiplexing capability for the simultaneous detection of multipleneuronal cell markers enables robust gene expression analysis and visualization of distinct cell populations within the nervous system (see Image Gallery). Furthermore, our BaseScope™ assay goes beyond gene expression detection by allowing for the detection of specific exon-exon junctions in alternatively spliced transcripts in the tissue environment. Alternative splicing is especially prominent in the brain, generating much of the complexity of the brain transcriptome.

"RNAscope® has streamlined in situ RNA hybridization to the point where it can become a standard technique easily implemented in most labs, even by individuals with no experience with this often difficult technique. Moreover the ability to multiplex ISH to simultaneously analyze the mRNA levels of up to three different genes allowed the study of changes in gene expression in specific cell populations in the CNS in response to different experimental conditions. Also this technique has been useful to analyze mRNA sub-cellular localization, which may prove important to understand disease mechanisms in neurodegenerative diseases”.

Miguel Sena-Esteves, PhD, Associate Professor, University of Massachusetts Medical School

Neuroscience Applications of the RNAscope® assay:

Detection, characterization and (co-) localization of mRNAs in the central and peripheral nervous systems including cell type-specific and subcellular target localization

The RNAscope® assay can be used for visualization of multiple target co-expression patterns or the co-expression of the target(s) of interest with desired cell type markers that characterize particular types of neurons or glia. Commonly used cell type markers to distinguish neuronal and glial target expression patterns include Rbfox3, Aif1, and Gfap for the detection of neurons, microglia and astrocytes, respectively (figure 1 in image gallery). The striatum harbors two distinct neuronal populations that either express Dopamine Receptor D1 (Drd1) – the striatonigral pathway – or Dopamine Receptor D2 (Drd2) - the striatopallidal pathway (figure 2 in image gallery). These different neuronal cell types can be accurately detected and visualized using the RNAscope® technology. Images for the detection of these different cell type markers can be found in our image gallery.

In a paper regarding research for perineuronal nets in hippocampal CA2 neurons, Carstens et al. J Neurosci 2016 (1) used the RNAscope® assay to demonstrate the co-localization of Aggrecan mRNA – a perineuronal net component - and Pcp4 mRNA - a marker of hippocampal CA2 neurons. Moreover, ISH results were consistent at the protein level for co-localization of Aggrecan and Pcp4 protein.

The single-molecule resolution of RNAscope® technology coupled with morphological context allows for the sub-cellular localization of mRNA expression in the cell body, axons or dendrites. Using the RNAscope® assay to confirm RT-PCR findings, Gervasi et al. RNA 2016 (2)provided the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated in the distal axons of SCG neurons.

Detection of long non-coding RNAs (lncRNAs) in the central and peripheral nervous systems

Recent studies have suggested the involvement of lncRNAs in neurodevelopment and brain function. Despite their functional importance, the mechanisms by which lncRNAs control cellular processes are still elusive. RNAscope® technology can shed light on the brain-enriched lncRNA functionality by visualizing tissue-restricted expression patterns, localization of lncRNAs to distinct subcellular structures, and regulated expression. Examples for the detection of lncRNAs NEAT1 and MALAT1 can be found in our image gallery (figure 3).

The RNAscope® technology can be used to validate gene expression profiling studies or RT-PCR results. Lake et al. Science 2016 (3) used both RNAscope® chromogenic and multiplex fluorescent ISH for a set of selected markers and confirmed subtype-and layer-specific expression patterns in the cortex revealed by single-cell transcriptomics for the human brain. Quantitative analysis showed that gene expression levels determined by the RNAscope® assay RNAs were consistent with RNA-seq data. Greer et al. Cell 2016 (4) employed the RNAscope® technology to validate RNA profiling expression data and interrogate the expression pattern of 12 members of the Ms4a family in necklace sensory neurons.

Validation of (cell type-specific) genetic modifications

RNAscope® ISH can be used to validate knock-out models or transgene expression patterns. In their study investigating the influence of the orphan GPCR Gpr88 in A2AR-expressing Drd2 neurons on anxiety-like behaviors, Meirsman et al. eNeuro 2016 (5) used multiplex fluorescent RNAscope® ISH for Gpr88, Drd1 and Drd2 to verify the specific excision of Gpr88 in striatal Drd2-medium spiny neurons of the conditional A2AR-driven Gpr88 knock-out. Seidemann et al. eLife (6) reports results of a new method using the genetically encoded calcium indicator GCaMP6f for chronic imaging of neural population responses in the primary visual cortex V1 of behaving monkeys. The RNAscope® Multiplex Fluorescent assay was used for the spatial analysis and validation of virus-mediated GCaMP expression in tissue samples from a GCaMP6f expressing site in a macaque 10 weeks after viral injection. They showed that 80% of cells were CaMKIIa-positive and all neurons expressing GCaMP were CaMKIIa-positive (attesting to promotor fidelity).

Detection of mRNA in the nervous system when no (reliable) antibodies are available

IHC is a well-established method for a broad range of applications from discovery to diagnostic and prognostic tests. However, raising antibodies to G-protein coupled receptors (GPCRs) has been challenging due to problems in obtaining suitable antigen accessibility since GPCRs are often expressed at low levels in cells and are very unstable when purified (Hutchings et al. 2010 (7)). Ion channels, another class of membrane proteins, also constitute a challenging class of targets for antibody discovery since they must remain membrane-associated to maintain their native conformation. RNAscope® technology is an ideal method to visualize these targets within their morphological context in the central and peripheral nervous systems. Examples for the detection of GPCRs and ion channels can be found in our image gallery (figure 4 to 8).

Visualization of neuronal network activity and plasticity

RNAscope® ISH can be applied for the visualization of activity and plasticity markers in the brain by means of detecting immediate early genes. Immediate early genes (IEGs) are genes which are transiently and rapidly activated at the transcriptional level in response to a wide variety of stimuli without the need for de novo protein synthesis (Guzowski et al.(8) 2002). The indirect detection of neuronal activity in the mouse brain hippocampus is shown by using a probe for Cfos which is a proto-oncogene and transcription factor. To detect neuronal plasticity, the expression of the effector immediate early gene Activity-Regulated Cytoskeleton-associated protein Arc (also known as Arg3.1) can be mapped. Images for these two markers can be found in the image gallery (figure 9 and 10).

By checking this box, I agree to receive relevant marketing communications regarding products, industry news, literature, and events from Bio-Techne and its brands:Advanced Cell Diagnostics, R&D Systems, Novus Biologicals, Tocris Bioscience, and ProteinSimple. I understand that I can change my preferences at any time. I further understand that any data provided to Bio-Techne and its brands will be stored, used, and deleted consistent with Bio-Techne’s Privacy Notice

Contact Name

Email

Phone

Company

Sample Type:

Product Line:

Description

Type the characters that you see in the picture below

By checking this box, I agree to receive relevant marketing communications regarding products, industry news, literature, and events from Bio-Techne and its brands:Advanced Cell Diagnostics, R&D Systems, Novus Biologicals, Tocris Bioscience, and ProteinSimple. I understand that I can change my preferences at any time. I further understand that any data provided to Bio-Techne and its brands will be stored, used, and deleted consistent with Bio-Techne’s Privacy Notice